1. Field of the Invention
The present invention relates to the use of lasers to mediate inactivation of infectious agents, such as viruses, bacteria, fungi and protozoans.
2. Description of the Background Art
There are a variety of viral inactivation approaches known in the art. For example, heat treatments and organic solvents have been used to conduct viral inactivation. There are drawbacks, however. The treatments can denature or inactivate the important biologically active substances, such as a blood proteins. Additionally, the use of substances like organic solvents can result in toxicity problems, and therefore such substances must be removed during the pharmaceutical finishing process.
As an alternative, light-based inactivation approaches have been proposed and are now achievable. Certain approaches rely on ultraviolet light.
The use of ultraviolet light began in the early part of the 20th century when it was recognized that mercury-based UV lamps had a germicidal effect on surface microorganisms. Commercially available UV technology has evolved and has resulted in a widespread use of UV light technology for a variety of commercial applications based upon new generations of reliable, higher power UV lamps.
For example, U.S. Pat. No. 5,364,645 discloses the use of monochromatic low energy pulsed ultraviolet laser irradiation with pulse duration in the range of picoseconds to microseconds at 1-1900 mJ/cm.sup.2 to inactivate microorganisms in food. This patent, however, has no applicability to the production of pharmaceuticals, particularly those based upon and/or containing proteins.
U.S. Pat. No. 4,880,512 concerns biological media such as blood fractions, plasma or genetically engineered protein products, that are treated to inactivate viruses. The proteins are kept substantially intact. According to example 2 of the patent, plasma is treated at 258 nm with a flux of 10.sup.15 photons/cm.sup.2 using mercury lamp technology. The bacteriophage T4 titer was reduced by 10.sup.6 while the protein activity has remained at 65% of its original value. According to example 3, 90% of the plasma protein activity remained after a flux of 10.sup.17 photons/cm.sup.2. In example 4, the treatment of factor VIII with 10.sup.14 photons/cm.sup.2 to inactivate T7 by a factor of 10.sup.6 is described and a remaining protein activity of 98%. However, the technique employed was a combined ultraviolet and visible light treatment. Additionally, flux of 10.sup.17 photons/cm.sup.2 corresponds to a high energy level of about 800 J/cm.sup.2.
U.S. Pat. No. 4,871,559 concerns food products that are preserved by inactivating microorganisms and/or enzymes by applying pulses of near visible light. The inactivation of enzymes, however, means that the approach in the '559 patent would deleteriously affect proteins.
WO 94/28120 concerns improving irradiation treatments by using ultraviolet light additives to quench to photodynamic reactions. For example, factor VIII recovery is increased in the presence of a quencher, such as unsaturated fatty acids, reduced sugars and indole derivatives. According to example 11, factor VIII recovery after ultraviolet irradiation was less than 50% in the absence of added quenchers. This publication, however, teaches reliance on the addition of substances like quenchers, which can interfere with the inactivation process.
Although not bound by any theory, it is thought that UV-mediated inactivation occurs through the structural changes it imparts to polynucleotides, such as those found in the genome of the pathogen. For example, UV radiation can cause the formation of thymine dimers between adjacent thymine molecules found in the genome. The formation of thymine dimers can prevent the replication of DNA. UV also can deleteriously impact DNA and RNA by causing hydration of pyrimidine bases.
Despite the above effects, the art still does not accomplish an effective and efficient laser-mediated inactivation approach. Methodologies that result in high viral titer inactivation require high energy and often denature the proteins found in solution. To minimize denaturation, substances like protein stabilizers (such as amino acids) and quenchers can be added, but these substances can decrease the level of inactivation. To increase the efficacy of inactivation while using lower energy levels, photodynamic substances (for example, methylene blue) can be employed. Photodynamic substances, however, are considered contaminants when making a pharmaceutical preparation, and therefore must be removed.
These problems remained unsolved until the development of the present invention.